Compositions for promoting wound healing and treating psoriasis

ABSTRACT

The present invention relates to compositions and methods for promoting healing of cutaneous, mucosal and/or mucocutaneous lesions associated with the presence of a mycoplasma and one or more non-mycoplasma microorganisms. The compositions and methods of the invention also relate to the reduction of joint pain, column pain, and/or skeletal muscle pain

PRIORITY CLAIM

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/952,003, filed Sep. 28, 2004, which is acontinuation-in-part of International Patent Application No.PCT/BR03/0049, filed Mar. 28, 2003 and published in English on Oct. 9,2003 as WO 03/082324 (priority to both Ser. No. 10/952,003 andPCT/BR03/0049 being claimed herein), and further claims priority to U.S.Provisional Application Ser. No. 60/890,980, filed Feb. 21, 2007, allthree of which are incorporated by reference in their entireties herein.

INTRODUCTION

The present invention relates to compositions and methods for promotinghealing of cutaneous, mucosal and/or mucocutaneous lesions by topicalapplication of a protein capable of removing sialic acid residues and aplant extract comprising nucleic acid containing particles.

BACKGROUND

Mycoplasmas represent some of the smallest self-replicatingmicroorganisms, and have unique properties among the prokaryotes. Theseproperties include the need for cholesterol to maintain their membraneenvelope, and the absence of an external wall. Mycoplasmas are known tocause pulmonary infection in humans, and it is widely known thatmycoplasmas can cause disease in most animals including humans as wellas animals of commercial importance such as cattle, swine, and fowl.(Razin et al., 1998, Microbiol. and Molecular Biology Review,62(4):1094-1156; Maniloff et al. Eds., 1992, Mycoplasmas, MolecularBiology and Pathogenesis, American Society for Microbiology,Washington).

Frequent co-occurrence of mycoplasma with other microorganisms, such aschlamydia, has been observed in diseases involving cell proliferation(International Patent Application No. PCT/BR01/00083, filed Jul. 3, 2001and BR PI 0002989-0, filed Jul. 3, 2001). This association appears toincrease the virulence of both pathogens. Mycoplasmal lipoproteins arepotent macrophage activators and have a comparable activity anddistribution in mollicutes as the LPS of Gram-negative bacteria. (Razinet al. Eds., 2002, Molecular biology and pathogenicity of mycoplasmas,Kluwer Academic/Plenum Publishers, New York). Recently describedtoll-like receptors (TLR) in macrophages that are activated by productsfrom pathogens such as mycoplasmal lipoproteins and LPS from bacteriahave been demonstrated to be important for activation of the immunesystem and it appears that the efficacy of the immune response dependson which concomitant TLR s are activated. (Akira et al., 2001, NatureImmunol. 2:675-680).

Archaea are the most ancient microorganisms existing in nature, but havebeen characterized only recently. See, Woese et al., Proc Natl. Acad.Sci. U.S.A. 74: 5088-5090 (1977). They inhabit extreme environments andare constituted by lipid monolayer membranes. Rich alkaline atmospherewith sodium ions and metals prevents proliferation of other bacteria,but is favorable to archaea's growth. Archaea have been isolated fromalkaline waters from the Dead Sea, the Great Salt Lake and YellowstoneNational Park. They have a small size, can—just barely—be viewed with anoptical microscope, and observation of structural details requireselectron microscopy. See, Howland et al., The surprising archaea.Discovering another domain of life, Oxford University Press (New York,2000). Some are considered hyperthermophilic as they survive in veryhigh temperatures.

Another unusual characteristic of some archaea is that they appear touse metal as an energy source. See, Amend et al., F.E.M.S. Microbiol.Rev. 25: 175-243 (2001). It is considered that archaea usually need ananaerobic or nearly anaerobic environments to carry outoxidation-reduction reactions with participation of different chemicalcompounds, including metals.

Recently, a new kind of extremely small archaea, which is dependent onbigger archaea, was described and named nanoarchaea. See, Huber Jet al.,Nature 417: 63-67 (2002). With the exception of archaea that reside inthe mammalian intestine and produce methane gases, there is no report ofarchaea existing within plants or animals. See, Florin T H J et al., Am.J. Gastroenterol. 95: 2872-2879 (2000).

Cutaneous lesions, for example, psoriasis and radiodermatitis resultingfrom radiotherapy, present obstacles to successful wound healing, as thelesions result from continued exposure to causative factors such asradiation therapy for cancer, or immune system dysfunction in psoriasis.In particular, the persistence of radiodermatitis lesions often requiresthe suspension of radiation therapy during cancer treatment in order topermit healing of the lesions. Such interruptions can be detrimental tothe successful treatment of cancer.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for promotinghealing of cutaneous, mucosal and/or mucocutaneous lesions by topicalapplication of a protein capable of removing sialic acid residues and aplant extract comprising nucleic acid containing particles. In variousembodiments of the invention, the composition is a topical gel, cream,or ointment comprising a protein capable of removing sialic acidresidues, such as a neuraminidase enzyme and/or a trans-sialidaseenzyme, and one or more purified plant extracts. In additionalembodiments, the composition further comprises a metal chelator.Administration of the topical formulation to the lesion promoteshealing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-F shows a decrease in edema and redness of radiodermatitis skinlesions following treatment with a trans-sialidase gel and an orchidextract gel. FIG. 1A-C show three different patients presentingradiodermatitis degree 4, represented by inflammation and cutaneousulcers (arrow) in a woman suffering from breast cancer (A), a woman withmalignant melanoma (B) and a man with larynx cancer (C). FIG. 1D-F showthe skin lesions of the same patients described in (A-C) following oneweek of treatment with trans-sialidase and orchid extract gels (arrow).The trans-sialidase gel was administered topically once per day to thelesions in an amount of 1.0 ml/50 cm² of cutaneous tissue each morningof treatment. The orchid extract gel was administered topically once perday to the lesions in an amount of 1.0 ml/50 cm² of cutaneous tissueeach evening of treatment. The

FIG. 2A-D shows lesions in a 59 year old woman suffering from psoriasisof the scalp before (A-B) and following (C-D) topical treatment withtrans-sialidase and orchid extract gels. (A-B) show psoriatic lesions(arrows) characterized by silvery scales on bright red, well-demarcatedplaques on the scalp. The plaques were usually associated with hairloss. (C-D) show the same lesions as in A-B following two months oftreatment with trans-sialidase and orchid extract gels. Thetrans-sialidase gel was administered topically once per day to thelesions in an amount of 1.0 ml/50 cm² of cutaneous tissue each morningof treatment. The orchid extract gel was administered topically once perday to the lesions in an amount of 1.0 ml/50 cm² of cutaneous tissueeach evening of treatment.

FIG. 3 shows the nucleotide sequence of a plasmid encoding the catalytictrans-sialidase unit of trans-sialidase from Trypanosoma cruzi (SEQ IDNO:3). The letters in capital represent the pET14b plasmid and theunderlined letters correspond to the position of the oligonucleotidesused to amplify the Trypanosoma cruzi clone.

FIG. 4 shows the amino acid sequence of the protein encoded by thenucleic acid sequence depicted in FIG. 3.(SEQ ID NO:4). In bold are theamino acids not found in the original clone.

FIG. 5 shows small dark electron-dense nanoarchae of between 0.03-0.15μm in diameter.

FIG. 6 shows dark medium sized electron-dense archaea of between 0.5-1.1μm in diameter, and large clear, empty archaea of between 1.0-2.4 μm indiameter.

FIG. 7 shows clear, empty archaea of between 0.15-2.0 μm in diameter.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of clarity, and not by way of limitation, the detaileddescription of the invention is divided into the following subsections:

(i) compositions for treating lesions; and

(ii) methods of treatment.

Compositions for Treating Cutaneous Lesions

The present invention provides for compositions and methods that promotethe healing of wounds, in particular, cutaneous, mucosal, and/ormucocutaneous lesions, Specifically, it has been found that thetreatment of lesions with compositions of the invention promotes thehealing of the lesions believed to be due to, without being bound to anyparticular theory, a decrease in the association of a mycoplasma and/orone or more non-mycoplasma microorganisms with the lesion being treated.

In non-limiting embodiments of the invention, the composition comprisesa carrier, such as a gel, cream, ointment, or lotion which furthercomprises a protein capable of removing sialic acid residues, such as aneuraminidase enzyme, a trans-sialidase enzyme, or a combinationthereof, and/or one or more purified plant extracts, and optionally, ametal chelator. According to the invention, application of thecomposition to a lesion inhibits or prevents the attachment of amycoplasma and/or a non-mycoplasma microorganisms to the host cells.

The term “lesion” as described herein means any structural disturbance,defect or abnormality to the cuticle, skin, mucosa, or mucocutaneoustissues of a subject. Lesions include, for example, but not limited to,structural defects, for example, lacerations and surgical wounds,radiodermatitis or injury resulting from exposure to radiation,psoriasis, rashes, moles, cysts, pimples, warts, burns, irritations,abrasions, baldness, seborreic keratosis, keloid scars, chronicdermatitis, fibrosis, sclerosis, cutaneous thickening, scars, cutaneousdiscontinuities, and ulcers.

The term “composition,” as used herein means one or more agents orcombinations thereof effective to promote the healing of a lesion and/ordecrease the presence of a mycoplasma and non-mycoplasma microorganismwith the lesion. In one embodiment, the composition inhibits the abilityof the mycoplasma and non-mycoplasma to associate with a substrate, forexample, but not limited to, a lesion. In another non-limitingembodiment, the composition inhibits the association of a mycoplasma anda non-mycoplasma microorganism.

The term “carrier,” as used herein means any agent the composition ismixed with to facilitate topical administration of the composition to asubject. Examples of carriers include, but is not limited to, gels,creams, ointments, liquids, or powders. In a preferred embodiment, thecarrier is a gel.

The term “mycoplasma” as used herein means a microorganism of the genusMycoplasma such as, but not limited to, Mycoplasma (M.) buccale, M.faucium, M. fermentans, M. genitalium, M. hominis, M. lipophilum, M.oral, M. penetrans, M. pneumoniae, M. salivarium, or M. spermatophilum.The one or more additional non-mycoplasma microorganism may be abacteria, archaea or virus, for example, but not limited to, spirocheteor chlamydia such as Chlamydia pneumoniae. According to the invention,the mycoplasma and non-mycoplasma may be attached to a substrate, forexample, but not limited to, a lesion. In a further non-limitingembodiment, the mycoplasma and non-mycoplasma are attached to thesubstrate by sialic acid.

In a preferred embodiment of the invention, the protein capable ofremoving sialic acid residues is a trans-sialidase or neuraminidaseenzyme.

In certain non-limiting embodiments, the composition comprises aneuraminidase enzyme of, for example but not limited to, Bacteroidesfragilis, Streptococcus pneumoniae, Streptococcus oralis, Arthrobacterureafaciens, Clostridium perfringens, Mycoplasma alligatoris,Arcanobacterium pyogenes, Clostridium sordellii, Pseudomonas aeruginosa,Micromonospora viridifaciens, Vibrio cholerae. Streptomyces avermitilis,Influenza virus, Streptomyces coelicolor, Flavobacteriales bacterium,and Solibacter usitatus.

In another non limiting embodiment, the protein comprises atrans-sialidase, for example, the trans-sialidase enzyme of Trypanosomabrucei.

In a preferred embodiment of the invention, the composition comprisesthe trans-sialidase enzyme of Trypanosoma cruzi, or a portion or variantof the native enzyme which has trans-sialidase activity.

Alternatively, the trans-sialidase enzyme may be a recombinanttrans-sialidase enzyme.

According to the invention, the recombinant trans-sialidase is asdescribed in International Patent Publication WO/2002/002050 by Higuchiet al., published Jan. 10, 2002; and U.S. Pat. No. 7,108,851 by Higuchiet al., issued Sep. 19, 2006. For example, the trans-sialidase gene maybe obtained from a genomic clone, isolated from a commercially availablelambda Zap® II library (Stratagene, http://www.stratagene.com) of T.cruzi Y strain (Silva and Nussenzweig, 1953, Folia Clin Biol 20:191-203), as described in Uemura et al. (Uemura et al., 1992, EMBO J 11:3837-3844). From the original lambda clone, which expresses enzymaticactivity, an SK plasmid containing the trans-sialidase gene may begenerated (SK-154-0). The preferred plasmid used is pTSII, whichcorresponds to a fragment of the original gene (clone 154-0) amplifiedthrough PCR, and inserted into the sites Ndel and BamHl of the vectorpET14b (Novagen—http://www.novagen.com). The PCR product may beamplified using SK-154-0 as a template with the following primers:

(SEQ ID NO: 1) a) TSPET14: 5′-GGAATTCCATATGGCACCCGGATCGAGC (SEQ ID NO:2) b) RT154: 5′-CGGATCCGGGCGTACTTCTTTCACTGGTGCCGGT

The resulting PCR product should have a nucleic acid sequence as setforth in FIG. 3 (SEQ ID NO:3), and a corresponding amino acid sequenceas depicted in FIG. 4 (SEQ ID NO:4). The resulting plasmid may betransformed into the Escherichia coil BLB21 DE3. The construct can bemade in two steps due to an internal BamHl site in the trans-sialidasegene. The PCR product may be treated with BamHl and Ndel enzymes, andthe resulting fragments fractionated by electrophoresis on an agarosegel. The separated fractions may then be purified from the gel with theSephaglass purification kit (Amersham-Pharmacia). The 5′ Ndel-BamHldigestion fragment may be ligated into the pET14b vector which has beenpre-digested with BamHl and Ndel. The ligation products may be used totransform K12 DH5a E. coil cells. The plasmid containing E. coil cellsmay be selected and the plasmid purified by methods known in the art.The purified construct may be treated with BamHI, shrimp alkalinephosphatase, and ligated with the BamHI-BamHI-3′ fragment purified fromthe fractionation gel. The ligation products may then be used totransform K12 DH5a E. coli cells, from which clones expression oftrans-sialidase may be selected and purified. The final plasmid may beconfirmed by restriction analysis and used to transform the BLB21 DE3pLys strain of E. coil, from which recombinant trans-sialidase enzymecan be purified, as described in International Patent PublicationWO/2002/002050 by Higuchi et al., published Jan. 10, 2002; and U.S. Pat.No. 7,108,851 by Higuchi et al., issued Sep. 19, 2006.

Alternatively, the trans-sialidase enzyme may be purified from a cultureof Trypanosoma cruzi, such as, for example, a culture according toKloetzel et al. (Kloetzel et al., 1984, Rev. Inst. Med. Trop. SaoPaulo., 26:179-85). Supernatant from the culture may be filtered througha 1 μm pore filter in a vacuum chamber. The enzyme may be furtherpurified by filtering the supernatant through a 0.22 μm filter and thenprecipitating the filtrate with a 50% (NH₄)2SO₄ solution. Theprecipitates may then be dialyzed against phosphate-buffered saline, andpassed through a tresyl-agarose column comprising an immobilizedanti-trans-sialidase monoclonal or polyclonal antibody. The column maybe washed with phosphate-buffered saline, followed by an additional washwith 10 mM sodium phosphate, pH 6.5. The trans-sialidase may then beeluted with a 3.5 mM MgCl₂, 10 mM sodium phosphate, pH 6.0 solution. Thefractions eluted from the column may be filtered through a Sephadex G-25column equilibrated with 20 mM Tris-HCl, pH 8.0, to remove the MgCl₂.The trans-sialidase may be further purified by passage through a Mono Qcolumn equilibrated in 20 mM Tris-HCl, pH 8.0, and eluted with a lineargradient from 0 to 1 mM NaCl in the same buffer.

The purified enzyme derived from the culture should comprise 400 kDamultimeric aggregates. The enzymatic activity of the purifiedtrans-sialidase may be measured according to methods described inInternational Patent Publication WO/2002/002050 by Higuchi et al,published Jan. 10, 2002; and U.S. Pat. No. 7,108,851 by Higuchi et al.,issued Sep. 19, 2006.

In non-limiting embodiments, the purified trans-sialidase has anenzymatic activity of between 0.1 and 10 U/ml, more preferably between1.0 and 5.0 U/ml, and most preferably 1.3 U/ml.

In a further non-limiting embodiments, the plant extract may be derivedfrom, for example but not limited to, Allium sativum (garlic), Ginkgobiloba, tomato, orchid, guava, ginseng, for example Pfaffia paniculata(Brazilian ginseng); tobacco; or Zingiber officinale (ginger), whereinthe orchid is preferably of the genus Cymbidium, for example, yellow orgreen orchids from the genus Cymbidium (Cymbidium ssp.). Alternatively,the orchid is of the genus Dendrobium, for example, Dendrobium nobile orDendrobium moschatum.

The extract from plants may be obtained by adding a solvent, such as,for example, alcohol, to the plant tissue, for example, but not limitedto, roots, cloves, flower petals, or leaves which may be chopped, ormacerated prior to mixture with the solvent. The solvent may be mixedwith the plant tissue in a proportion of between 1:99 and 60:40, morepreferably between 15:85 and 50:50 and most preferably between30-40:70-60 of plant mass:alcohol. The solvent can be an alcohol, forexample, ethanol, methanol, or grain alcohol, and can have aconcentration of between 60% and 100%, more preferably between 70% and95%, and most preferably about 92% alcohol. The plant/alcohol mixturemay be aged in a dark, anaerobic environment for a period of timebetween 15 days and 24 months, more preferably between 1 and 12 months,and most preferably 10 months.

According to the invention, the extract derived from plant comprisesparticles containing nucleic acid (DNA and/or RNA), wherein the particleis an archaea (preferably non-pathogenic) and/or a nanoarchaea, andfurther wherein the particle is present in an amount effective toprevent or inhibit the growth of a mycoplasma and one or morenon-mycoplasma microorganisms. Aging of the plant/alcohol mixtureincreases the concentration of particles in the mixture.

The plant/alcohol mixture may be purified, and the concentration ofnanoparticles may be increased through one or more filtrations. Themixture can be filtered through pores of between 0.5 μm and 50 μm, morepreferably between 5 μm and 20 μm, and most preferably 11 μm, forexample, but not limited to Whatman qualitative filter paper grade 1,diameter 24 cm, pore size 11 μm. Vacuum chambers can also be usedseparately, or in addition to other filtration methods. Additionally,glass microfiber filters can be used, for example, but not limited to, a47 mm diameter glass microfiber filter with a pore size of 1.1 μm. Manfiltration methods are known in the art can be used to filter the agedplant/alcohol mixture.

In a non-limiting embodiment, the plant/alcohol mixture can be subjectedto additional aging during the filtration process. For example, oliveoil may be added to the filtrate to create a 1% olive oil filtratemixture, followed by an additional month of storage in a dark anaerobicenvironment.

Furthermore, the composition may comprise particles and/or nanoparticlescontaining DNA or RNA, wherein the particles are a non-pathogenicarchaea and/or a nanoarchaea, and further wherein the particle ispresent in amounts effective to prevent or inhibit the growth of amycoplasma and one or more non-mycoplasma microorganisms. Thenanoparticles may be between 5-500 nm, more preferably between 15-250nm, and most preferably between 30-150 nm in diameter. Alternatively,the composition may comprise medium particles of between 500 nm and 1.1μm in diameter. Additionally, the compositions may comprise one or acombination of both small and medium particles. The size of a particlecan enlarge or decrease depending on the concentration of water and ionsin a solution comprising the particles, such as, for example, Na+ orCa+.

According to the invention, the purity of the plant extract may bedetermined by microscopic examination of the filtered, aged, plantextract, as described in U.S. Patent Application Publication No.20050142116. For example, the filtered, aged plant extract can bestained with any DNA or RNA dye known in the art, such as acridineorange, bisbenzimide H 33342 (Hoechst), or4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI); and viewed withan immunofluorescence optical microscope, an electron microscope, or anyother microscope known in the art. Two forms of archaea, havingdifferent morphological characteristics may be identified. One typecomprising an electron-dense content may be between about 0.03-0.15 μm(nanoparticle) and about 0.5-1.1 μm in diameter (medium particle) (FIGS.5 and 6, respectively). A second type may comprises a clear, emptycontent, and may be about 0.15-2.4 μm in diameter (FIGS. 6 and 7). Theclear, empty archaea are similar in morphology to the pathogenic archaeaassociated with lesions, while the electron dense archaea comprise thenon-pathogenic archaea and nanoarchaea comprising DNA or RNA. Brilliantred particles, which may comprise metallic ions, may also adhere to thesurface of the archaea. Optimum purity may be achieved whenpredominantly, preferably essentially, only fast moving electron-densenanoparticles are visible. The presence of clear, empty archaea or largebrilliant red particles of about 0.15-0.24 μm and at a concentration of,for example, ≧1.0 large brilliant red particle/visual field, indicatessuboptimal purity. In cases of suboptimal purity, the filtered agedplant extract is subjected to additional filtration, for example,tangential flow filtration in the Minitan Ultrafiltration System(Millipore, Bedford, Mass., USA), using the microporous membrane packet(30,000 NMWL). In preferred embodiments, the compositions of theinvention comprise a greater number of electron dense archaea(nanoparticles and medium particles) than empty, clear archaea; and agreater number of archaea not associated with large brilliant redparticles than those associated with large brilliant red particles.

According to the invention, the purified plant extract may comprise anenriched population of particles. The concentration of particles may bebetween 1×10⁵ and 1×10¹⁰ particles/ml, more preferably between 1×10⁶ and1×10⁹ particles/mi, and most preferably about 1×10⁷ particles/ml.

In certain non-limiting embodiments, the composition comprises a metalchelator, for example, but not limited to, Nitrilotriacetate (NTA),diphenylthiocarbazone(dithizone), histidine, the lipophilic metalchelator DP-109, ethylene glycol tetraacetic acid (EGTA),ethylenediaminetetraacetic acid (EDTA), DMPS(2,3-dimercapto-1-propanesulfonate), Lysinoalanine, Syntheticlysinoalanine (N-ε-DL-(2-amino-2-carboxyethyl)-L-lysine), tetracycline,alpha lipoic acid (ALA), Dimercaptosuccinic acid, (DMSA),2,3-Dimercapto-1-propanesulfonic acid (DMPS), Calcium disodium versante(CaNa₂-EDTA), D-penicillamine, Deferoxamine, Defarasirox, Dimercaprol(SAL), the calcium salt of diethylene triamine pentaacetic acid (DTPA),or any other metal chelator known in the art. In a preferred embodiment,the metal chelator is pyrrolidine dithiocarbamate (PDTC). In a preferredembodiment, the metal chelator is pyrrolidine dithiocarbamate (PDTC).The composition of the invention may comprise the metal chelator in aconcentration of between about 0.01 and 10 mg/ml, more preferablybetween about 0.5 and 5 mg/ml, more preferably between about 1 and 2mg/ml, and most preferably about 1.5 mg/ml.

In a non-limiting embodiment, the compositions of the invention comprisecombinations of trans-sialidase, a metal chelator, and one or morepurified plant extracts as shown in Table I.

TABLE I Combinations of trans-sialidase, a metal chelator, and one ormore purified plant extracts encompassed by the invention. Combinationsof trans-sialidase (TS), pyrrolidine dithiocarbamate (PDTC), andpurified plant extracts TS TS + PDTC TS + PDTC + Allium sativum (AS)TS + PDTC + Ginkgo biloba (GB) TS + PDTC + Zingiber officinale (ZO) TS +PDTC + orchid extract (OE) TS + PDTC + AS + GB TS + PDTC + AS + ZO TS +PDTC + AS + OE TS + PDTC + AS + GB + ZO TS + PDTC + AS + GB + OE TS +PDTC + AS + GB + ZO + OE TS + PDTC + AS + ZO + OE TS + PDTC + GB + ZOTS + PDTC + GB + OE TS + PDTC + GB + ZO + OE TS + PDTC + ZO + OE TS + ASTS + GB TS + ZO TS + OE TS + AS + GB TS + AS + ZO TS + AS + OE TS + AS +GB + ZO TS + AS + GB + OE TS + AS + GB + ZO + OE TS + AS + ZO + OE TS +GB + ZO TS + GB + OE TS + GB + ZO + OE TS + ZO + OE

Methods of Treatment

The present invention provides for compositions and methods that promotethe healing of wounds, in particular, cutaneous, mucosal, and/ormucocutaneous lesions. Specifically, it has been found that thetreatment of lesions with compositions of the invention promotes thehealing of the lesions believed to be due to, without being bound to anyparticular theory, a decrease in the association of a mycoplasma and/orone or more non-mycoplasma microorganisms with the lesion being treated.In a non-limiting embodiment of the invention, the composition may beadministered topically to a lesion as a lotion, cream, liquid, paste,ointment, powder, or any other carrier known in the art. In a preferredembodiment of the invention, the composition is applied as a topicalgel.

In one non-limiting embodiment, the composition of the invention isapplied in an amount effective to promote healing of wounds, inparticular, cutaneous, mucosal, and/or mucocutaneous lesions, forexample, but not limited to, structural defects, for example,lacerations and surgical wounds, radiodermatitis or injury resultingfrom exposure to radiation, psoriasis, rashes, moles, cysts, pimples,warts, burns, irritations, abrasions, baldness, seborreic keratosis,keloid scars, chronic dermatitis, fibrosis, sclerosis, cutaneousthickening, scars, cutaneous discontinuities, and ulcers.

The term “treatment” as defined herein means a reduction in lesion size,inflammation, irritation, scaling, scar formation, fibrosis, sclerosis,and cutaneous thickening. In a non-limiting embodiment, the treatmentmay reduce the presence of moles, cysts, pimples, warts, burns,irritations, abrasions, baldness, seborreic keratosis, keloid scars,chronic dermatitis, cutaneous discontinuities, structural defects,lacerations and surgical wounds.

In a non-limiting embodiment, the methods and compositions of theinvention are effective for treating a lesion, wherein topicaladministration of the composition may be effective to reduce the size ofthe lesion by at least about 1%, 5%, 10%, 25%, 50%, 75%, 90%, or 100%.

The present invention provides for formulations, to be applied topicallyto a cutaneous, mucous, or mucocutneous lesion. The composition maycomprise between about 1×10⁻⁸ and 1×10⁻⁴ U/ml, or between about 1×10⁻⁷and 1×10⁻⁵ U/ml, or between about 1'10⁻⁶ and 5×10⁻⁶ U/ml, and mostpreferably about 2×10⁻⁶U/ml of neuramidase and/or trans-sialidaseactivity. The composition may also comprise between about 100 and 1×10⁷plant extract derived particles/ml, or between about 1×10³ and 1×10⁶particles/ml, or between about 1×10⁴ and 5×10⁵ particles/ml and mostpreferably about 3×10⁵ particles/ml. Optionally, the composition maycomprise a metal chelator, for example, but not limited to, PDTC, NTA,diphenylthiocarbazone(dithizone), histidine, DP-109, EGTA, EDTA, DMPS,Lysinoalanine, Synthetic lysinoalanine, tetracycline, ALA,Dimercaptosuccinic acid, DMSA, Calcium disodium versante,D-penicillamine, Deferoxamine, Defarasirox, Dimercaprol, or DTPA, in aconcentration of between about 0.01 and 10 mg/ml, or between about 0.5and 5 mg/ml, or between about 1 and 2 mg/ml, and most preferably about1.5 mg/ml.

In specific non-limiting embodiments, the composition comprises 2.6×10⁻⁶U/ml neuramidase and/or trans-sialidase activity, and/or 3×10⁵ plantderived particles/ml.

Alternatively, the composition may comprise between about 0.01 and 10U/ml, or between about 0.2 and 5 U/ml, or between about 0.5 and 2 U/mland most preferably about 1.0 U/ml of neuramidase and/or trans-sialidaseactivity.

In one embodiment of the invention, the gel is a trans-sialidase gel,wherein the trans-sialidase gel is created by mixing trans-sialidaseenzyme with a gel, for example, but not limited to, a non-ionic,anionic, or cationic gel. In one preferred embodiment, the gel is ahydroxyethylcellulose gel, for example, Natrosol® gel Thetrans-sialidase enzyme may be diluted with a suitable dilutant, forexample, but not limited to, MilliQ purified water, distilled water, orthermal water, prior to mixing with the carrier. In a preferredembodiment, the dilutant is MilliQ purified water. When diluted, thetrans-sialidase is diluted at a trans-sialidase:dilutant ratio ofbetween 1:1 and 1:2,000,000, more preferable between 1:100 and1:1,000,000, more preferably between 1:1000 and 1:500,000, and mostpreferably 1:250,000. The resulting trans-sialidase dilution, oralternatively, undiluted enzyme, may then be mixed with a gel at atrans-sialidase:gel ratio of between 1:1000 and 1000:1, more preferablybetween 1:100 and 100:1, more preferably between 1:10 and 10:1 and mostpreferably 1:1.

In one, non-limiting example, the trans-sialidase gel is made bydiluting a purified trans-sialidase enzyme 1:250,000 in purified water,for example, MilliQ purified water. The diluted trans-sialidase is thenmixed into Natrosol® gel (Dermavita, Brusque, Santa Catarina, Brazil),at a concentration of 1:1 and stored at 4° C. The gel may be appliedtopically to a subject in an amount of 1.0 ml/50 cm² of cutaneoussurface.

In one non-limiting embodiment of the invention, the trans-sialidaseenzymatic activity of the trans-sialidase gel is between 100 and 100,000CPM, more preferably between 1000 and 20,000 CPM, more preferablybetween 1,500 and 10,000 CPM, and most preferably between 2,000-5,000CPM, wherein 30,000 CPM corresponds to 0.36 nmoles of transferred sialicacid in a 30 min assay, which is further defined as one (1) unit (U).

In a non-limiting embodiment of the invention, the trans-sialidase gelmay be administered topically to a subject in need of treatment, whereinthe gel is administered one or more times per day. For example, thetrans-sialidase gel may be administered once, twice, three, four, five,or 6 or more times per day. In a non-limiting example, thetrans-sialidase gel may be applied topically once per day in the morningor evening during treatment.

In another non-limiting embodiment, the gel is a purified plant extractgel, preferably an orchid extract gel. The purified plant extract may bediluted with a suitable dilutant, for example, but not limited to,MilliQ purified water, distilled water, or thermal water, prior tomixing with the carrier. When diluted, the purified plant extract isdiluted at a plant extract:dilutant ratio of between 1:100 and 100: 1,more preferably between 1:50 and 50:1, more preferably between 1:10 and10:1, and most preferably 1:5. The diluted or undiluted purified plantextract may be mixed with a gel, for example, but not limited to, ananionic gel. In a preferred embodiment, the gel is ahydroxyethylcellulose gel, for example, Natrosol® gel.

In a further non-limiting embodiment, the gel and the diluted orundiluted purified plant extract are mixed to achieve a gel with a finalplant extract concentration of between 0.1% and 99%, more preferablybetween 5% and 80%, more preferably between 10% and 50%, and mostpreferably 15%.

In a non-limiting example, the purified plant extract is diluted 1:5 inthermal water (e.g. from Irai-RS, Brazil, or another thermal watersource), which was previously boiled and filtered. The diluted plantextract is then mixed with an anionic gel (Dermavita, Brusque, SantaCatarina , Brazil; or another thermal water source) until the mixtureachieved a final concentration of 15% plant extract. The gel may beapplied typically to a subject in an amount of 1.0 ml/50 cm² ofcutaneous surface.

In a non-limiting embodiment of the invention, the purified plantextract gel may be administered topically to a subject in need oftreatment, wherein the gel is administered one or more times per day.For example, the purified plant extract gel may be administered once,twice, three, four, five, or 6 or more times per day. In a non-limitingexample, the purified plant extract gel may be applied topically onceper day in the morning or evening during treatment.

In another non-limiting embodiment of the invention, both the purifiedplant extract gel and the trans-sialidase gel may be administeredtopically to a subject in need of treatment, wherein the gels areadministered one or more times per day, for example, the purified plantextract gel and the trans-sialidase gel may be administered once, twice,three, four, five, or 6 or more times per day. In a further non-limitingembodiment, the purified plant extract gel and the trans-sialidase gelmay be administered simultaneously or in series. In a non-limitingexample, the trans-sialidase gel may be applied topically once per dayin the morning and the purified plant extract gel may be appliedtopically once per day in the evening during treatment. Alternatively,the gels may be applied topically at the same time.

In an additional non-limiting embodiment of the invention, thecomposition comprises both trans-sialidase and one or more purifiedplant extracts, for example, a gel comprising both a trans-sialidase geland a purified plant extract gel. The two gels may be combined toproduce a gel with a final trans-sialidase gel:purified plant extractgel ratio of between about 99:1 and 1:99, more preferably between 90:10and 10:90, more preferably between 85:15 and 15:85, and most preferably80:20.

The gel mixture comprising both the trans-sialidase gel and the purifiedplant extract gel may comprise a trans-sialidase enzymatic activity ofbetween about 1×10⁻⁸ and 1×10⁻⁴ U/ml, or between about 1×10⁻⁷ and 1×10⁻⁵U/ml, or between about 1×10⁻⁶ U/ml, and preferably about 2×10⁻⁶ U/ml.The gel mixture may further comprises between about 100 and 1×10⁷ plantextract derived particles/ml, or between about 1×10³ and 1×10⁶particles/ml, or between about 1×10⁴ and 1×10⁵ particles/ml andpreferably about 6×10⁴ particles/ml.

In a specific, non-limiting embodiment, the gel comprises both atrans-sialidase enzymatic activity of 2.08 U/ml and 6×10⁴ plant derivedparticles/ml.

In a non-limiting embodiment, the gel comprising both trans-sialidaseand one or more purified plant extracts may be administered topically toa subject in need of treatment, wherein the gel is administered one ormore times per day. For example, the gel may be administered once,twice, three, four, five, or 6 or more times per day. In a non-limitingexample, the gel may be applied topically once per day in the morning orevening during treatment.

In a non-limiting embodiment of the invention, the composition isapplied to a lesion in an amount between about 0.1 ml/50 cm² and 100ml/50 cm², or between about 0.5 ml/50 cm² and 50 ml/50 cm², and mostpreferably about 1.0 ml/50 cm² of cutaneous surface.

In an alternative embodiment, the composition is applied to the lesionin an amount between about 0.1 ml/5 cm² and 100 ml/5 cm², or betweenabout 0.5 ml/5 cm² and ml/5 cm², and most preferably about 1.0 ml/5 cm²of cutaneous surface.

In a non-limiting example, the methods and compositions of the inventionmay be effective for treating radiodermatitis lesions. Daily topicaladministration of the trans-sialidase gel once per day in the morning,and the purified plant extract gel once per day in the evening during atreatment period which may be between one week, two weeks, one month,two months, or three months and one year may be effective to reduce thelesions. The gels may be administered in an amount of between about 1.0ml/5 cm² and 1.0 ml/50 cm², or between about 1.0 ml/5 cm² and 50 ml/50cm², or between about 1.0 ml/5 cm² and 100 ml/50 cm², wherein thetrans-sialidase gel comprises an enzymatic activity of between about1×10⁻⁸ and 1×10⁻⁴ U/ml, or between about 1×10⁻⁷ and 1×10⁻⁵ U/ml, orbetween about 1×10⁻⁶ and 5×10⁻⁶ U/ml, and preferably about 2.6×10⁻⁶U/ml. The purified plant extract gel may comprise between about 100 and1×10⁷ plant extract derived particles/ml, or between about 1×10³ and1×10⁶ particles/ml, or between about 1×10⁴ and 5×10⁵ particles/ml andpreferably about 3×10⁵ particles/ml. Alternatively, the gel may be amixture of both the trans-sialidase gel and the purified plant extractgel wherein the gel mixture comprises a trans-sialidase enzymaticactivity of between about 1×10⁻⁸ and 1×10⁻⁴ U/ml, or between about1×10⁻⁷ and 1×10⁻⁵ U/ml, or between about 1×10⁻⁶ and 5×10⁻⁶ U/ml, andpreferably about 2.08×10⁻⁶ U/ml, and further wherein the gel mixturecomprises between about 100 and 1×10⁷ plant extract derivedparticles/ml, or between about 1×10³ and 1×10⁶ particles/ml, or betweenabout 1×10⁴ and 1×10⁵ particles/ml and preferably about 6×10⁴particles/ml.

The treatment may be administered between intervals of radiationtreatment for cancer. Such treatment may be effective to reduce the sizeof radiodermatitis lesions by about 1%, 5%, 10%, 25%, 50%, 75%, 90%, or100%.

In another non-limiting example, the methods and compositions of theinvention may be effective for treating psoriasis. Daily topicaladministration of the trans-sialidase gel once per day in the morning,and the purified plant extract gel once per day in the evening during atreatment period which may be between one week, two weeks, one month,two months, or three months and one year may be effective to reduce thepsoriatic lesions. The gels may be administered in an amount of betweenabout 1.0 ml/5 cm² and 1.0 ml/50 cm², or between about 1.0 ml/5 cm² and50 ml/50 cm², or between about 1.0 ml/5 cm² and 100 ml/50 cm², whereinthe trans-sialidase gel comprises an enzymatic activity of between about1×10⁻⁸ and 1×10⁴ U/ml, or between about 1×10⁻⁷ and 1×10⁻⁵ U/ml, orbetween about 1×10⁻⁶ and 5×10⁻⁶ U/ml, and preferably about 2.6×10⁻⁶U/ml. The purified plant extract gel may comprise between about 100 and1×10⁷ plant extract derived particles/ml, or between about 1×10³ and1×10⁶ particles/ml, or between about 1×10⁴ and 5×10⁵ particles/ml andpreferably about 3×10⁵ particles/ml. Alternatively, the gel may be amixture of both the trans-sialidase gel and the purified plant extractgel wherein the gel mixture comprises a trans-sialidase enzymaticactivity of between about 1×10⁻⁸ and 1×10⁻⁴ U/ml, or between about1×10⁻⁷ and 1×10⁻⁵ U/ml, or between about 1×10⁻⁶ and 5×10⁻⁶ U/ml, andpreferably about 2.08×10⁻⁶ U/ml, and further wherein the gel mixturecomprises between about 100 and 1×10⁷ plant extract derivedparticles/ml, or between about 1×10³ and 1×10⁶ particles/ml, or betweenabout 1×10⁴ and 1×10⁵ particles/ml and preferably about 6×10⁴particles/ml.

The treatment may be administered between intervals of radiationtreatment for cancer. Such treatment may be effective to reduce the sizeof radiodermatitis lesions by about 1%, 5%, 10%, 25%, 50%, 75%, 90%, or100%.

Examples Example 1 Treatment of Radiodermatitis and Inflamed CutaneousLesions

In the following study, thirty-one patients presenting severe skinlesions caused by radiodermatitis after radiotherapy were treated withdaily topical application of a trans-sialidase gel and an orchid gel tothe lesions. Application of the gels decreased the edema and redness ofthe radiodermatitis lesions.

Materials and Methods Production of Purified Plant Extract

Orchid flowers, Cymbidium ssp., Dendrobium nobile and Dendrobiummoschatum were immersed whole in 92% ethanol, in a 30:70 proportionplant weight:ethanol. The mixture was stored in a dark, anaerobicenvironment (in a sealed bottle), for at least 10 months. Followingstorage, the mixture was passed through Whatman qualitative filter papergrade 1, diameter 24 cm, pore size 11 urn. Olive oil was then added at avolume of 10 ml per 1000 ml of the filtrate and stored for at least oneadditional month to increase the concentration of nanoparticles in themixture. The mixture was then filtered a second time in Whatmanqualitative filter paper grade 1. The filtrate was next filtered in avacuum chamber, with a 47 mm diameter glass microfiber filter, pore sizeof 1.1 μm.

To determine the purity of the extract, 10 μl of the extract was mixedwith 5 μl of acridine orange and placed on a glass microscope slide. Themixture was examined on an immunofluorescence optical microscope at 400×magnification. The extract quality was considered optimum when only fastmoving small nanoparticles (30-150 nm) were visible. If large brilliantred particles (0.15-0.24 um) were observed in the mixture at aconcentration of ≧1.0 large particle/visual field, the extract wassubmitted to tangential flow filtration in the Minitan UltrafiltrationSystem (Millipore, Bedford, Mass., USA), using the microporous membranepacket (30,000 NMWL) that concentrates large particles. The filtrate wasthen used in the experiments.

Production of Orchid Gel

The purified orchid extract was diluted 1:5 in thermal water (fromIrai-RS, Brazil), which was previously boiled and filtered. The dilutedorchid extract was then mixed in an anionic gel (Dermavita, Brusque,Santa Catarina, Brazil) until the mixture achieved a final concentrationof 15% orchid extract.

Production of Trans-Sialidase

Purified trans-sialidase was produced from the Escherichia coli BLB21DE3 inserted with the pTSII plasmidium, as described previously(International Patent application no. PCT/BR01/00083, filed Jul. 3,2001).

Production of Trans-Sialidase Gel

Pure recombinant trans-sialidase was diluted 1:250,000 in MilliQpurified water. The diluted trans-sialidase was then mixed intoNatrosol® gel (Dermavita, Brusque, Santa Catarina, Brazil), at aconcentration of 1:1 and stored at 4° C. The trans-sialidase enzymaticactivity of the gel mixture was between 2,000-5,000 CPM.

Production of a Trans-Sialidase-Orchid Gel Mixture.

A combined mixture of the orchid extract gel and trans-sialidase gel wascreated by mixing the two together. The two gels were combined toproduce a final gel mixture that was 20% orchid extract gel and 80%trans-sialidase gel, or was in an orchid extract gel:trans-sialidase gelration of 1:1, 1:2.5, 1:6 or 1:20.

Treatment with Orchid Extract Gel and Trans-Sialidase Gel

Thirty one patients presenting severe skin lesions including edema andredness, in the most severe cases associated with ulcers, caused byradiodermatitis after radiotherapy (median 5040 cGy; radiodermatitisdegree 2-4) were treated with orchid extract gel and trans-sialidasegel. Treatment with the gels was administered between intervals ofradiotherapy. Patients received treatment with the gels for a one yearperiod. 26 patients were receiving radiotherapy for malignant neoplasia,and 5 patients were receiving radiotherapy for different inflammatorycutaneous discontinuities (Table II). Patients were treated with dailytopical application (1.0 ml/50 cm2 of cutaneous surface) oftrans-sialidase gel (once in the morning) and orchid gel (once in theevening). An additional 7 patients presenting radiodermatitis grade 3-4lesions received treatment with a combined orchid extract gel andtrans-sialidase gel during the treatment period.

Results

The 7 patients treated with the combined mixture of orchid extract geland trans-sialidase gel, and 30 of the 31 patients treated with the oncedaily applications of the trans-sialidase gel and the orchid extract gelseparately exhibited a decrease of edema and redness within two to eightdays of treatment, along with healing of ulcers (FIG. 1). Untreatedlesions require between at least 20-30 days for healing to occur. Onepatient who was receiving chemotherapy in combination with radiotherapyduring the study did not exhibit a decrease in edema or redness.

TABLE II Clinical data of the 31 patients treated with trans-sialidasegel and orchid extract gel. Radiodermatitis Case Sex/age Diagnosisdegree 1. F/50 y Breast cancer 2 2. F/42 Breast cancer 2 3. F/54 Breastcancer 2 4. F/63 Breast cancer 3 5. F/69 Breast cancer 3 6. F/62 Breastcancer 2 7. M/32 Melanoma 2 8. F/58 Breast cancer 2 9. M/54 Larynxcancer 3 10. F/38 Melanoma 4 11. F/62 Breast cancer 3 12. F/45 Breastcancer 3 13. M/67 Rectum cancer 4 14. F/64 Breast cancer 2 15. F/38Breast cancer 3 16. F/49 Breast cancer 2 17. F/28 skin dehiscence — 18.F/28 Breast cancer 3 19. F/46 Breast cancer 4 20. F/63 Breast cancer 221. F/50 Breast cancer 3 22. F/70 Breast cancer 2 23. F/84 Breast cancer2 24. F/58 Breast cancer 2 25. F/57 Thymoma 3 26. F/50 Breast cancer 227. F/62 foreign body reaction — 28. M/62 fistulae — 29. M/61 skindehiscence — 30. F/51 Legs chronic dermatitis — 31. M/71 Chemotherapyulcers —

Example 2 Treatment of Psoriasis and Psoriatic Arthritis withTrans-Sialidase Gel and Orchid Extract Gel

A 59 years old woman with severe psoriasis of the scalp, as well as manyother parts of the body, was treated with the trans-sialidase and orchidextract gels. The two gels were prepared as described previously. Thepatient was treated by topical application of the gels once per day fortwo months. The trans-sialidase gel was applied in the morning, and theorchid extract gel was applied at night during each day of treatment.Following treatment, the psoriasis of the scalp regressed, exhibiting adecrease in the presence of the skin lesions (FIG. 2).

A 47 year old woman with severe psoriatic arthritis received treatmentwith the trans-sialidase and orchid extract gels, prepared as previouslydescribed. The psoriatic arthritis presented as inflammation of thejoints associated with psoriasis of the scalp, arms, hands, and legs.The psoriatic arthritis was characterized by retraction andimmobilization of the fingers, and difficulty in raising the arms.Immunohistochemical analysis performed on cutaneous biopsies aspreviously described in Higuchi et al., 2006, APMIS 114:338-344,revealed a high presence of Mycoplasma pneumoniae and Chlamydiapneumoniae antigens on the affected tissue. The patient was treated forone year with both trans-sialidase and orchid extract gels. Each gel wasapplied once per day with the trans-sialidase gel applied in themorning, and the orchid extract gel applied at night. Following one yearof treatment, the patient experienced a decrease in the presence of skinlesions, and an increase in mobility of the fingers and arms.

Example 3 Treatment of Joint and Column Pain with Trans-Sialdase Gel andOrchid Extract Gel

Ten patients suffering from joint and column pain, which was associatedwith articulation inhibition, were treated with the orchid extract gelor a mixture of both the orchid extract gel and the trans-sialidase gelfor more than one year.

Three patients that were at least 65 years of age topically applied theorchid extract gel each time joint or column pain was experienced.Treated patients reported pain relief to be immediate, or within onehour after treatment with the orchid gel, and the relief lasted at leastone day.

Seven patients aged 35 to 65 years with articular and muscle pain due toarthritis of different etiologies, bursitis and idiopathic myalgia, weretreated with a combined orchid extract gel and trans-sialidase gel mixedat a ration of 1:1. The combined gel was applied twice a day (once everytwelve hours) during a 15-30 day treatment period. Pain relief occurredin less than one hour following treatment and persisted during theentire treatment period.

Various publications are cited herein, the contents of which are herebyincorporated by reference in their entireties.

1-7. (canceled)
 8. A composition for promoting healing of a lesioncomprising an agent that can remove sialic acid residues and one or moreplant extracts comprising nucleic acid-containing particles selectedfrom the group consisting of archaea, nanoarchaea, and a mixturethereof.
 9. The composition of claim 8, wherein the composition isapplied topically.
 10. The composition of claim 8, further comprising acarrier selected from the group consisting of a gel, cream, ointment,and lotion.
 11. The composition of claim 8, further comprising a metalchelator.
 12. The composition of claim 11, wherein the metal chelator ispyrrolidine dithiocarbamate (PDTC).
 13. The composition of claim 8,wherein the compound that can remove sialic acid residues is an enzymeselected from the group consisting of trans-sialidase, neuraminidase,and a combination of a trans-sialidase and a neuraminidase.
 14. Thecomposition of claim 8, wherein the plant extract is an extract from aplant selected from the group consisting of garlic, ginkgo, tomato,orchid, guava, ginseng, ginger, and tobacco.